The inherent structural diversity of carbohydrates poses a major analytical challenge to all aspects of the glycoscience and is one reason why glycomics lags behind the advances that have been made in genomics and proteomics. The structure of a carbohydrate is described by its composition, connectivity, and configuration (see FIG. 1). The carbohydrates or glycans are composed essentially of monosaccharides (e.g. D-heptoses, L-heptoses, D-hexoses, L-hexoses, D-pentoses, L-pentoses, D-tetroses, L-tetroses, sialic acids), which are connected to each other via glycosidic linkages (e.g. α-(1→2), β-(1→2), α-(1→3), β-(1→3), α-(1→4), β-(1→4), α-(1→5), β-(1→5), α-(1→6), β-(1→6)). The composition of a carbohydrate (I) is defined by the monosaccharides (e.g. Glc, Man, Gal, Fuc) that are forming said carbohydrate. These building blocks are often stereoisomers that differ merely in the stereochemistry at one particular carbon atom, as in the case of glucose (Glc) and galactose (Gal). Each monosaccharide contains multiple hydroxyl groups that can be a point of attachment for a glycosidic linkage with the next monosaccharide. Thus, unlike oligonucleotides and proteins, carbohydrates are not necessarily linear, but rather can be branched structures with diverse regiochemistry (II). Additionally, a new stereocenter emerges when a glycosidic bond is formed, because two monosaccharides can be connected in two different configurations (III), thus leading to α and β anomers.
Carbohydrates structure is typically ascertained by a combination of nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). Measuring a mass-to-charge ratio (m/z) with MS is fast, requires very little sample and provides precise, high-resolution data about the sample composition. Detailed information regarding connectivity can be obtained following derivatisation and/or elaborate tandem MS analysis. Nevertheless, with MS it is not possible to analyze stereoisomers, since they generally cannot be distinguished due to their identical atomic composition and mass.
NMR experiments serve best to determine configurational information of carbohydrates, but require large amounts of sample, are time consuming and the resulting spectra are cumbersome to interpret when different stereoisomers need to be distinguished. In addition, the relative detection limit of 3-5% for larger oligosaccharides in NMR experiments is rather poor. Liquid chromatography (LC) can help to differentiate configurational isomers, but an unambiguous identification of one isomer in the presence of another is often not possible either.
To overcome the above-mentioned limitations, it is the objective of the present invention to provide a method for determining in an expedient manner and with minimal sample consumption the structure of a target carbohydrate by using ion mobility-mass spectrometry (IM-MS) in negative ionization mode. Herein, the negative ions of the carbohydrates, as well as the fragments of the negative ions of the carbohydrates are not only separated according to their mass and charge, but also based on their size and shape, thereby providing information about the underlying three-dimensional structure. IM-MS measures the drift time i.e. the time that ions require to drift through a cell that is filled with an inert neutral gas, such as helium or nitrogen, under the influence of a weak electric field. During this, compact ions undergo fewer collisions with the gas than more extended ions, and therefore traverse the cell faster. The measured drift time can be converted into an instrument-independent, rotationally averaged collision cross section (CCS) by methods known to the skilled person in the art (Anal. Chem. 2013, 85, 5138-5145; Anal. Chem. 2014, 86, 10789-10795). The rotationally-averaged collision cross-section (CCS) represents the effective area for the interaction between an individual ion and the neutral gas through which it is travelling. The CCS is intrinsic to a particular carbohydrate and is influenced by both the ionic state (i.e. positive/negative mode and adduction), as well as the particular drift gas.
The objective of the present invention is solved by the teaching of the independent claims. Further advantageous features, aspects and details of the invention are evident from the dependent claims, the description, the figures, and the examples of the present application.